According to statistics published on the homepage of the American Cancer Society, cancer accounts for nearly one-quarter of deaths in the United States, exceeded only by heart disease. In 2000, there were 553,091 cancer deaths in the US. A number of nucleoside analogues are currently used to treat different types of cancers, HIV and other disorders. Some of these are shown in Table 1, below.
TABLE 1Anti-Cancer AgentsCommon ortrade nameCAS numberFormulaLamivudin134678-17-42′-deoxy-3′-thiacytidineZidovudine30516-87-13′-azido-3′-deoxythymidineZalcitabine7481-89-22′,3′-dideoxycytidineStavudine3056-17-52′,3′-didehydro-3′-deoxythymidinedidanosine69655-05-62′,3′-dideoxyinosine,Abacavir136470-78-52-Cyclopentene-1-methanol, 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-,(1S,4R)-(9CI)floxuridine50-91-95-fluoro-2′-deoxy uridinefludarabine21679-14-112-fluoro-9-b-D-arabinofuranosyladenineCytarabine147-94-41-B-D-arabinofuranosylcytosineazacitidine320-67-25-azacytidineDecitabine2353-33-55-aza-2′-deoxycytidineThioinosine22140-20-16-mercaptopurineriboside85-31-46-thioguanosinecladribine4291-63-82-chlorodeoxyadenosinepentostatin53910-25-12′ deoxycoformycinGemcitabine95058-81-42′-deoxy, 2′,2′-difluorocytosine
It has been shown recently that the human genome has highly conserved metameric GCn*GC motif clusters located across genes and promoter regions. Such GCn*GC sites are vulnerable genomic sites that are preferentially destroyed in both apoptotic and necrotic cell death commitment, with specific megabase DNA fragmentations revealed in pulsed-field gel electrophoretic data analysis. (Yee-Jiun Kok, Myint Swe, and Kwok-Hung Sit, Biochemical and Biophysical Communications, 294: 934-939, 2002).
Normal cells are euploid and do not have drug/multidrug resistance. Cancerous cells, on the other hand, have a continuously evolving aneuploid phenotype resulting from high genetic instability and vulnerability, which is associated with high mutational rates, and drug and multidrug resistance (PNAS 98: 11283-11288, 2001; PNAS 97: 14295-14300, 2000; Cancer Genet. Cytogenet. 119: 83-93, 2000; Nature Biotechnology 19: 22-23, 2000).
Untargeted genomic destruction by treatment with many anticancer drugs does cause cell death. Gemcitabine, for example, is most likely incorporated into DNA as a competitive inhibitor of deoxycytidine by the de novo DNA synthesis pathway. Gemcitabine is a prodrug, which most likely gets phosphorylated in the cell to be active. It has been shown to be a better permeant than cytrabine (arabinosylcytosine), another anticancer drug (Biochemical Pharmacology 46(4): 762-766, 1993). The nucleoside is a favorable substrate for deoxycytidine kinase, and thus accumulates in cells in greater amounts. Further, the formation of Gemcitabine triphosphate and its negative feedback regulation of deoxycytidine deaminase, results in favorable incorporation of Gemcitabine via its triphosphate, into DNA. The incorporation of Gemcitabine into DNA results in cell death. Gemcitabine has a vastly improved therapeutic index as compared to many anticancer drugs. However this cell death remains non-specific to cancerous cells in that death is induced equally in both cancerous and normal cells. Thus, although Gemcitabine (Gemzar) (like other anticancer drugs) is effective in causing cell death and is approved by the FDA for the treatment of a range of cancers, there is no differential killing between normal and cancer cells in non-targeted continuous treatment (See, e.g., Eli Lilly publication attached hereto as Appendix A).
There is therefore an urgent need for an approach in which selective targeting of potent anticancer drugs to cancer cells can be achieved.
It has been shown by recent studies (Biochem. Biophy. Res. Commun. 294: 934-939, 2002) that cell death, whether by necrosis or apoptosis, has orderly DNA fragmentation. Thus kilobase and 200 by DNA ladder fragmentations obtained by way of necrosis from freezing, demonstrated a marked pattern of DNA base sequence selection, similar to that in apoptosis. The genomic GC clusters with high density were preferentially GCn*GC (SEQ ID NO: 1) a motifs. They seem to align with metameric regularity in the genome of man to virus, and thus preserve a high degree of regularity in the chromatin conformation.
Necrosis from freezing manifested an orderly pattern of DNA fragmentations including the apoptosis signature of 200 by ladder, in three different cell populations despite pancanpase suppression by zVAD-fmk. Immediately on thawing, all three populations had 100% dead cell indices and 2.2, 1.6, and 1.1 megabase fragmentations, which marked the point of death. Kilobase and 200 by DNA ladder fragmentations manifested later together with overt necrotic morphologies. CpG oligodeoxynucleotides (ODNs) complementary to highly conserved GCnGC (SEQ ID NO: 2) motifs inhibited the megabase fragmentations and retarded their electrophoretic mobility (gel shift), indicating ODN-DNA binding, which is known to confer site-specific resistance to cleavage. Cleavage specificity was confirmed using EDTA-CpG ODN conjugates to direct free-radical-producing transitional element, vanadyl(4), to the binding sites to reproduce the megabase fragmentations in normal cells. Specific orderly fragmentation in necrosis suggested a necrosis-apoptosis convergence after death has been committed (Biochem. Biophy. Res. Commun. 294: 934-939, 2002).
Thus, in a recent study, it was shown that the complementary CGn*CG (SEQ ID NO: 3) oligodeoxynucleotide (ODN) sequences in parallel orientation were able to inhibit megabase DNA fragmentation, indicating ODN-DNA binding, and thereby conferring site-specific resistance to cleavage (Biochem. Biophy. Res. Commun. 294: 934-939, 2002). A series of ODN, with GCn*GC (SEQ ID NO: 1) motifs, where n=2, 5, 9, were synthesized in this study. The test examples synthesized were 5′-GCnnGC-3′ (SEQ ID NO: 4), 5′-GCnnnnnGC-3′ (SEQ ID NO: 5), 5′-GCnnn nnn nnn GC-3′ (SEQ ID NO: 6). These oligonucleotides, which are complementary to the highly conserved GCn*GC motifs, after conjugation inhibited the megabase fragmentations, thereby indicating ODN-DNA binding.
Few other CpG motifs are widespread in nature. It has been stated that certain CpG oligonucleotides stimulate strong, balanced immunity by boosting antibody based cellular responses to antigens presented by infectious pathogens or cancerous cells. Based on this approach a CpG oligonucleotide developed is currently on clinical trial, phase I/phase II, as a monotherapy or multidrug therapy for non-Hodgkins' lymphoma, basal cell carcinoma and melanoma (Eugene Uhlmann, in the Oligonucleotide and Peptide Conferences, May 6-8 Tides 2002, Las Vegas, Nev.).
Bacterial DNA has been shown to contain CpG DNA. CpG DNA has been shown to stimulate B-cell proliferation and activate macrophages, monocytes and dendritic cells. The activation of immune cells by CpG DNA results in the secretion of a number of cytokines, including IL-6, IL-12, TNF-a and TNF-r (Wagner, H. (2000) Immunology of Bacterial CpG-DNA, Springer-Verlag, Heidelberg, Germany; Raz, E. (2000) Immunostimulatory DNA sequences. Springer-Verlag, Heidelberg, Germany). Thus, divergent therapeutic and immunologic effects of oligonucleotides with CpG motifs have been reported (Ballas, Z. K., Krieg, A. M., Warren, T., Rasmussen, W., Davis, H. L., Waldschmidt, M. and Wagner, G. J., J. Immunol. 167, 4878-4886, 2001). CpG DNA has been similarly shown to overcome hyporesponsiveness to Hepatitis B vaccine in orangutans. (Davis, H. L., Suparto, I. I., Weeratna, R. R., Jumintarto, Iskandriati, D. D., Chamzah, S. S., Ma'ruf, A. A., Nente, C. C., Pawitri, D. D., Krieg, A. M., Vaccines, 18: 1920-1924, 2000). CpG oligonucleotides have been shown to trigger protective and curative ThI responses in lethal murine leishmaniasis (Zimmermann, S., Egeter, O., Hausmann, S., Lipford, G. B., Rocken, M., Wagner, H., Heeg, K. J. Immunol., 160: 3627-3630, 1998). More recently synthetic phosphorothiate oligonucleotides containing CpG dinucleotides were shown to possess immunostimulatory activity (Dong Yu, Ekamber R. Kandimalla, Qiuyan Zhao, Yanping Cong and Sudhir Agarwal, Nucleic Acids Res., 30: 1613-1619, 2002). Further, these authors reported novel 3′,3′-linked CpG oligodeoxynucleotides as potent immunostimulatory agents with key design and two or more identical CpG DNA segments (Dong Yu, Ekamber R. Kandimalla, Lakshmi Bhagat, Jin Yan Tang, Yanping Cong, Jimy Tang and Sudhir Agarwal, Nucleic Acids Res., 30: 4460-4469, 2002). In a recent study improved oligodeoxynucleotides (ODN) have been synthesized having a CpR motif, which have potent immunostimulatory properties, and cause increased induction of interleukin (IL)-12 and lesser secretion of IL-6 (Ekamber R. Kandimalla, Lakshmi Bhagat, Daqing Wang, Dong Yu, Fu-Gang Zhu, Jimmy Tang, Hui Wang, Ping Huang, Ruiwen, and Sudhir Agarwal, Nucl. Acids Res., 31: 2393-2400, 2003).